Saturated fatty acids are known to be the precursors of unsaturated fatty acids in higher organisms. However, the control mechanisms that govern the conversion of saturated fatty acids to unsaturated fatty acids are not well understood. The relative amounts of different fatty acids have an effect on the physical properties of membranes. Furthermore, regulation of unsaturated fatty acids is important because they play a role in cellular activity, metabolism and nuclear events that govern gene transcription.
A critical committed step in the biosynthesis of mono-unsaturated fatty acids is the introduction of the first cis-double bond in the delta-9 position (between carbons 9 and 10). This oxidative reaction is catalyzed by stearoyl-CoA desaturase (SCD, also known as delta-9-desaturase) and involves cytochrome b5, NADH (P)-cytochrome b5 reductase and molecular oxygen (Ntambi, J. Lipid Res., 1999, 40, 1549–1558). Although the insertion of the double bond occurs in several different methylene-interrupted fatty acyl-CoA substrates, the preferred substrates are palmitoyl- and stearoyl-CoA which are converted to palmitoleoyl- and oleoyl-CoA respectively (Ntambi, J. Lipid Res., 1999, 40, 1549–1558).
It has been recognized that, irrespective of diet, the major storage fatty acids in human adipose tissue are oleic acid, an 18 carbon unsaturated fatty acid, and palmitoleic acid, a 16 carbon unsaturated fatty acid (Ntambi, J. Lipid Res., 1999, 40, 1549–1558). During the de novo synthesis of fatty acids, the fatty acid synthase enzyme stops at palmitoleic acid but the end product of the pathway is usually oleic acid (Ntambi, J. Lipid Res., 1999, 40, 1549–1558).
The stearoyl-CoA desaturase gene was partially characterized in 1994 via isolation of a 0.76 kb partial cDNA from human adipose tissue (Li et al., Int. J. Cancer, 1994, 57, 348–352). Increased levels of stearoyl-CoA desaturase mRNA were found in colonic and esophageal carcinomas and in hepatocellular carcinoma (Li et al., Int. J. Cancer, 1994, 57, 348–352). The gene was fully characterized in 1999 and it was found that alternative usage of polyadenylation sites generates two transcripts of 3.9 and 5.2 kb (Zhang et al., Biochem. J., 1999, 340, 255–264). Two loci for the stearoyl-CoA desaturase gene were mapped to chromosomes 10 and 17 and it was determined that the chromosome 17 loci encodes a transcriptionally inactive pseudogene (Ntambi, J. Lipid Res., 1999, 40, 1549–1558).
Disclosed and claimed in PCT publication WO 00/09754 is a nucleic acid molecule encoding the human stearoyl-CoA desaturase and a nucleic acid molecule which, under suitable conditions, specifically hybridizes to said nucleic acid molecule encoding the human stearoyl-CoA desaturase (Stenn et al., 2000).
Stearoyl-CoA desaturase affects the ratio of stearate to oleate which, in turn, affects cell membrane fluidity. Alterations of this ratio have been implicated in various disease states including cardiovascular disease, obesity, non-insulin-dependent diabetes mellitus, skin disease, hypertension, neurological diseases, immune disorders and cancer (Ntambi, J. Lipid Res., 1999, 40, 1549–1558). Stearoyl-CoA desaturase has been viewed as a lipogenic enzyme not only for its key role in the biosynthesis of monounsaturated fatty acids, but also for its pattern of regulation by diet and insulin (Ntambi, J. Lipid Res., 1999, 40, 1549–1558).
The regulation of stearoyl-CoA desaturase is therefore of considerable physiologic importance and its activity is sensitive to dietary changes, hormonal imbalance, developmental processes, temperature changes, metals, alcohol, peroxisomal proliferators and phenolic compounds (Ntambi, J. Lipid Res., 1999, 40, 1549–1558).
Animal models have been very useful in investigations of the regulation of stearoyl-CoA by polyunsaturated fatty acids (PUFAs). For example, in adipose tissue of lean and obese Zucker rats, Jones et al. observed a 75% decrease in stearoyl-CoA desaturase mRNA when both groups were fed a diet high in PUFAs relative to a control diet (Jones et al., Am. J. Physiol., 1996, 271, E44–49).
Similar results have been obtained with tissue culture systems. In the murine 3T3-L1 adipocyte cell line, arachidonic linoleic, linolenic, and eicosapentanenoic acids decreased stearoyl-CoA desaturase expression in a dose-dependent manner (Sessler et al., J. Biol. Chem., 1996, 271, 29854–29858).
The molecular mechanisms by which PUFAs regulate stearoyl-CoA desaturase gene expression in different tissues are still poorly understood. The current understanding of the regulatory mechanism involves binding of PUFAs to a putative PUFA-binding protein, after which repression transcription occurs via binding of the PUFA-binding protein to a cis-acting PUFA response element of the stearoyl-CoA desaturase gene (SREBP) (Ntambi, J. Lipid Res., 1999, 40, 1549–1558; Zhang et al., Biochem. J., 2001, 357, 183–193).
Cholesterol has also been identified as a regulator of stearoyl-CoA desaturase gene expression by a mechanism involving repression of the maturation of the sterol regulatory element binding protein (Bene et al., Biochem. Biophys. Res. Commun., 2001, 284, 1194–1198; Ntambi, J. Lipid Res., 1999, 40, 1549–1558).
Thiazolidinediones have been employed as regulators of stearoyl-CoA desaturase activity in murine 3T3-L1 adipocytes (Kim et al., J. Lipid Res., 2000, 41, 1310–1316), and in diabetic rodents (Singh Ahuja et al., Mol. Pharmacol., 2001, 59, 765–773).
Disclosed and claimed in PCT publication WO 99/63979 are compositions comprising a saponin in an amount effective to inhibit stearoyl-CoA desaturase enzyme activity wherein said saponin is derived from a source selected from the group consisting of Quillaja saponaria, Panax trifolium, Panax quinquefolium and Glycyrrhiza glabra (Chavali and Forse, 1999).
Disclosed and claimed in European patent EP 238198 is an inhibitor of stearoyl-CoA desaturase prepared in a form suitable for oral, parenteral, rectal or dermal administration for use in modifying the lipid structure of cell membranes wherein said inhibitor consists of a saturated fatty acid having from 12 to 28 carbon atoms in the alkyl chain, e.g. stearic acid, or a pharmaceutically acceptable derivative thereof prepared in a form suitable for parenteral, rectal or dermal administration (Wood et al., 1987). A stearoyl-CoA desaturase antisense vector has been used to reduce expression levels of stearoyl-CoA desaturase in chicken LMH hepatoma cells (Diot et al., Arch. Biochem. Biophys., 2000, 380, 243–250).
To date, investigative strategies aimed at inhibiting stearoyl-CoA desaturase function include the previously cited use of polyunsaturated fatty acids, saturated fatty acids, thiazolidinediones, cholesterol, and an antisense vector. However, these strategies are untested as therapeutic protocols. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting stearoyl-CoA desaturase.
Antisense technology is emerging as an effective means of reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic and research applications involving modulation of stearoyl-CoA desaturase expression.
The present invention provides compositions and methods for modulating stearoyl-CoA desaturase expression, including inhibition of both isoforms of stearoyl-CoA desaturase.